Systematic review and meta-analysis on the use of human platelet lysate for mesenchymal stem cell cultures: comparison with fetal bovine serum and considerations on the production protocol

Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures. Graphical Abstract 1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com). Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-02815-1.


Background
To make mesenchymal stem cell (MSC)-based therapies a concrete clinical option, it is mandatory to implement the standardization process of the in vitro culture protocols starting from the first exploratory phases of the study. In fact, if the protocols comply with the regulatory requirements of cell-based therapy products, the evidences acquired during all the study phases can be easily translated into clinical application. In Europe, the manufacturing of MSC-based products for clinical purposes must be compliant with specific requirements set by the European Medicines Agency in the Regulation Recently, regulatory restrictions have been raised regarding the use of FBS in clinical settings, such as the risk of xeno-immunization against bovine antigens, transmission of pathogens, and ethical problems connected with the brutal procedures used for FBS collection [1]. Therefore, the interest towards human alternatives as medium supplements has grown fast to implement the bench-to-bedside translation of MSCbased therapies.

Graphical Abstract
1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com).
Page 3 of 31 Palombella et al. Stem Cell Research & Therapy (2022) 13:142 Among them, Human Platelet Lysate (HPL) has been proposed as substitute for FBS [1] given the platelet content rich in several bioactive molecules. Large amounts of HPL can be easily obtained from unused apheresis products and buffy coats, even in the form of pooled product from different blood donors, thus reducing inter-batch variability [2]. After the first description of the preparation and use of HPL for MSC expansion by Doucet and colleagues in 2005 [3], the use of HPL has been evaluated in several in vitro and pre-clinical studies on bone-marrow-(BMSCs) and adipose-derived (ASCs) mesenchymal stem cells [4]. The common trend among these studies is that HPL seems to support cell viability similarly to FBS, without altering other features of MSCs, such as immunophenotype, clonogenic ability, and genomic stability [5,6]. However, despite a common consensus regarding the potential of HPL, there is still large heterogeneity of production methods that inevitably leads to variable outcomes. The only common thread is the maximization of the release of growth factors by optimizing the platelet lysis [2]. Whereas, the relevance of a variety of factors possibly affecting the composition of HPL and therefore its efficacy, including donor's features, platelet source, and lysis method [1] is generally neglected. This lack of standardized recommendations and culture protocols partly prevents the scientific community from quickly and efficiently adopting HPL for culturing MSCs.
The aim of this meta-analysis and systematic review was therefore to systematically analyze the current literature that compares the effect of HPL and FBS on ASC and BMSC cultures. Differences between HPL and FBS were firstly evaluated by a meta-analysis to understand whether HPL influenced MSC proliferation differently than FBS, in terms of doubling time (DT) and population doubling (PD). Then, differences between these two supplements were evaluated qualitatively and described through the typical MSC features (i.e. clonogenic ability, immunophenotype, morphology, mesenchymal differentiation, and immunomodulatory properties). Moreover, the presence of a possible correlation between HPL production steps and cell proliferation rate of both ASCs and BMSCs was analyzed. Based on the collected results, considerations were made on the HPL production protocols as well as on strengths and weaknesses of the use of HPL in MSC culture.

Study design
This systematic review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist [7].

Search strategy
Literature search was performed by querying three different electronic databases: PubMed, The Cochrane Library, and Embase. Study selection was executed on articles published until the end of March 2021 and without other limitations on publication period. The keyword search was structured as following: "human platelet lysate" AND "fetal bovine serum" OR "fetal calf serum" OR "FBS" OR "FCS" AND "mesenchymal stem cell" OR "mesenchymal stromal cell" OR "stem cell" OR "stromal cell" OR "progenitor cell". The presence of all these terms was checked only within title or abstract. The full-texts of significant studies were further investigated to assess eligibility.

Study selection
Eligibility criteria were established a priori to include journal articles in the systematic review. The retrieved articles were first screened by title and abstract. Those that satisfied the following inclusion criteria were selected: (1) description of a direct comparison between HPL and FBS for the culture of BMSCs or ASCs; (2) 2D cell-culture on plastic only (e.g. not coated); (3) English language; (4) full-text availability.
Journal articles that (1) did not include the control group (FBS-cultured MSCs); (2) used pathogen-reduced HPL; (3) declared a previous MSC contamination with animal sera (e.g. collagenase inhibition with FBS prior to culture with HPL); (4) did not provide exhaustive information on cell isolation protocol; (5) employed different basal media between FBS-and HPL-cultured cells; and (6) changed the type of medium during cell culture were excluded from the analysis. Moreover, we did not consider conference abstracts, reviews, and duplicate published data.
In the second step, the full texts of the selected articles were screened, with further exclusions according to the previously described criteria. Two investigators evaluated all the articles independently. In case of disagreement, a third investigator was consulted.

Data extraction
Two investigators performed data extraction independently. For the included studies, relevant data were extracted from article texts, tables, and figures, and then summarized and analyzed to the purpose of the present work. In particular, the following data were collected for each study: (1) cell type (BMSCs and/or ASCs); (2) demographic features of donors; (3) isolation and expansion protocol; (4) percentage of FBS and HPL; (5) culture media and other supplements used (e.g. antibiotics and animal free trypsin); (6) proliferation rate and clonogenic/pluripotency ability; (7) immunophenotypic profile; (8) cell morphology; (9) tri-lineage mesenchymal differentiation potential; (10) safety properties (telomerase activity, karyotype analysis, etc.); (11) cell secretory activity; and (12) immunomodulatory potential. For all the analyzed records, only those experiments meeting the inclusion criteria were maintained.
When not clearly reported in the paper, numeric data were rigorously extracted using the online free software WebPlotDigitizer (by Ankit Rohatgi, https:// autom eris. io/ WebPl otDig itizer) with appropriate graph settings.

Outcomes
When culturing cells in vitro, the proliferation rate is one of the parameters that permits to evaluate the state of health of the cells. This parameter is easily calculable, objective, and allows to compare cell growing among different papers. Since the cell proliferation rate can be calculated indiscriminately as DT (the time a cell takes to double) or as PD (the number of times the cells double during the culture), we decided to consider both of them for the meta-analysis.
The primary outcome of this meta-analysis was the comparison of the proliferation rate expressed as (1) DT and (2) PD (cumulative and mean value) of cell cultured with FBS and HPL. The secondary outcome was qualitatively evaluated and performed on the following parameters: clonogenic ability (CFU-F assay), pluripotency (gene expression and immunofluorescence staining), immunophenotype (FACS), morphology (side and forward scatter, imaging), mesenchymal differentiation (staining, gene expression), cell safety (senescence, karyotype, telomerase activity), paracrine effect (ELISA assay), immunomodulatory abilities (functional assays with immune cell system).

Risk of bias (quality assessment)
Given the lack of an official checklist for the quality assessment of in vitro studies, we defined the risk of bias for the included studies starting from the existing checklist from Cochrane [8]. We defined specific questions related to main bias categories. For each study, we considered 4 categories as sources of bias with specific questions that could be used to evaluate the quality assessment of all in vitro studies. For each bias, "yes" or "no" was attributed accordingly to its presence or absence, respectively. Moreover, "unclear" was assigned when there the elements to judge were insufficient.

Data synthesis
Descriptive statistics were presented as median (interquartile range), mean (standard deviation) or number (percentage), depending on the analyzed parameter. Differences between FBS and HPL in terms of DT and PD from each single study were evaluated by meta-analysis subgrouping data according to the cell type, ASCs or BMSCs. Similarly, the effect of the percentage of HPL in culture medium (5% and 10%) was evaluated too. The meta-analysis was performed when outcome data were available from more than two studies, by using mean difference (MD) with 95% confidence intervals (CIs). Means were used as final values of each outcome adopting a random effects model since we expected high heterogeneity [9]. Heterogeneity was evaluated using the I 2 statistic [10] where I 2 > 75% is considered substantial heterogeneity. Thus, in the presence of substantial heterogeneity, we performed sensitivity analysis based on biological assumptions excluding high risk of bias related to missing or unclear methods used in the research practice (category 3-item 1) and inadequate number of cell donors (category 1-item 1). Data analyses were performed using RevMan Software 5.4, The Cochrane Collaboration, 2020. For hypothesis testing, a probability value of < 0.05 was considered as statistically significant. All statistical tests were 2-sided.
Associations between DT values and variables of HPL production protocols such as (1) the material used as platelet source (i.e. platelet apheresis, platelet rich plasma-PRP, buffy coat, whole blood), (2) the procedure used for the platelet lysis (less or more than 3 freeze/thaw cycles), and (3) the addition of heparin in the final culture medium were evaluated too. The association between the above-mentioned variables and DT was investigated with graphical tools. Statistical analysis was performed by GraphPad Prism v7.0 (Graphpad Software). Normality of data was assessed by Shapiro-Wilk tests. Wilcoxon matched pairs signed rank test was used to compare two conditions of the same parameter (e.g. heparin addition or not). Non parametric one-way (ANOVA) by Kruskal-Wallis test and Dunn's multiple comparisons test was used for comparison of more than two conditions (e.g. the type of starting material).

Study selection
The initial search identified 358 records. The study selection process led to the exclusion of 47 duplicate records and 5 articles written in a different language than English. Among the remaining records screened for title and abstract (n = 306), 6 reviews and 161 congress abstracts were excluded due to the lack of specific data and 70 because non-related records. The full-text of 69 articles was then analyzed. Among them, 36 articles that did not satisfy one or more inclusion criteria and 1 article that was a duplicate of another publication were excluded. Other 3 articles were retrieved from the bibliography of other papers, yielding a final number of 35 articles which were included in this review (Fig. 1).

Features of included studies
The main features of the studies included are reported in Tables 1 and 2. The first paper that directly compared FBS and HPL for BMSC culture was published in 2005, while the first paper concerning ASCs was published in 2009. BMSCs and ASCs were used in about 65.7% (23/35) and 28.6% (10/35) studies, respectively, whereas only two included both cell types [11,12]. In all the articles, 10% FBS in the culture medium was considered as the control group in this meta-analysis. Only one study described also the use of 5% and 20% FBS [13]. Conversely, the analyzed studies reported the use of various HPL concentrations. In particular, 10% and 5% HPL were applied in 23 and 16 studies, respectively. Other concentrations (0.5, 1, 2.5, 7.5, 8, 20%) were described in 7 studies articles only. Most papers used home-made HPL (31/35), whereas only 4 studies tested a commercial HPL (Table 1). Among the retrieved papers, two of them investigated the effect of autologous HPL on MSC expansion [14,15].
The enzyme collagenase was used in ASC-related studies for adipose tissue digestion (12/35). Two papers only   (Table 2).

Quantitative analysis of primary outcomes by meta-analysis
Most papers evaluated at least one parameter concerning cell proliferation between DT and/or PD (24/35) (Table 1). Overall, 22 studies reported data that were eligible for the meta-analysis. Among them, 9 articles reported data on ASCs and 13 on BMSCs. Data reported in one study were not considered for the metaanalysis because cells were expanded until they reached a specific PD, representing a diverging methodology from the other studies [19]. Among the studies included in the meta-analysis, only one applied autologous HPL and presented mediated results among donors [15]. The other paper using autologous HPL and presenting data divided for each donor was not considered [14].

Population doubling (PD)
Among the studies analyzed, 9 reported data regarding PD expressed as cumulative data measured at different passages. Table 3 reports the pooled analysis with available data at each passage (e.g. P1, P2, etc.) for the following comparisons: (a) FBS 10% versus HPL 10%, (b) FBS 10% versus HPL 5%. For the comparison HPL 10% versus HPL 5% insufficient information was available to provide pooled analyses. The forest plots of metaanalyses are reported as Additional file 1. Overall HPL, irrespective of its percentage in the medium (i.e. 10% or 5%), showed better results than FBS 10% albeit a very substantial heterogeneity (I 2 > 90%) and paucity of data (Table 3). Finally, 3 studies reported data regarding PD expressed as mean values among passages, of which 2 analyzed BMSCs [23,43] and 1 analyzed ASCs [24]. However, no pooled analyses were performed due to the paucity of information.

Descriptive analysis of MSC features related to medium supplementation CFU-F assay
The CFU-F assay was performed in 13/35 of the studies ( Table 1). Most of the papers (9/13) showed similar results between cells cultured in FBS or HPL in terms of number of colonies, whereas three studies [27,34,37] observed a greater number of colonies in cells cultured with HPL, and one paper [24] the opposite. More uniform data concerned the morphology of the cells forming the colonies and the size of the colonies themselves. Colony cells grown in HPL generally appeared smaller with spindle-shaped morphology and characterized by fast proliferation, indicating that the use of HPL promotes the formation of bigger colonies more densely packed with smaller cells, not observed in FBS cultures.

Stem cell markers expression
Only 2 papers investigated cell pluripotency by evaluating the expression of NANOG, OCT4, and SOX2 in ASCs and BMSCs [17,28]. Although both studies reported an increased expression of these genes in HPL-cultured cells compared to FBS, the immunofluorescence analysis revealed that the related proteins were expressed at the same level in cells cultured with FBS and HPL.

Immunophenotype
The immunophenotype analyzed by FACS was reported in 31/35 of the papers (Table 1). Overall, the results showed that the supplementation with HPL did not affect the expression of MSC-related markers compared to FBS culture, neither in ASCs nor in BMSCs. In fact, CD73, CD90, and CD105 were positively expressed (> 90%) whereas CD14, CD31, CD34, CD45, and HLA-DR were not or scarcely expressed (< 5%) by all groups, regardless of the culture supplement or cell type. The expression of other MSCs markers, such as CD19, CD29, CD44, and HLA-ABC, was reported only in one third of the articles and therefore it was not possible to sum up the data relative to their expression to conduct a quantitative analysis (Additional file 2).

Morphology
Cell morphology was described in 23/35 of the papers (Table 1). With the exception of one record [41], all the studies provided phase contrast pictures of cells cultured with both supplements. In general, HPL-cultured cells displayed a smaller and more elongated morphology, characterized by lower granularity compared to FBS-cultured cells. These features were confirmed by the decrease of forward and side scatter values analyzed with FACS in 2 papers [18,41]. Only one study reported that HPL triggered larger and more granular cells compared to FBS-cultured cells [24]. Six papers did not report any morphologic difference due to the different culture supplements.

Multi-differentiation potential
The ability to differentiate toward mesenchymal lineages was assessed in 26/35 of the studies (Table 1). Both BMSC and ASC achieved an efficient adipogenic, osteogenic and chondrogenic differentiation level, regardless of the use of HPL or FBS. Adipogenic differentiation was performed in 19/35 papers. Only 2 papers investigated the expression of adipogenic markers in ASCs [35,37], no one in BMSCs In particular, ADIPOQ and FABP4 resulted overexpressed [35] and PPARg downregulated in HPL-cultured cells [35,37]. Noteworthy, 2/19 papers (1 investigating ASCs and 1 BMSCs) were in countertrend and reported a lower number of differentiated cells in HPL-cultures compared to FBS-ones [32,37]. Remarkable, 1/19 paper investigated the effect of HPL from single donors on adipogenic differentiation and revealed that the level of differentiation was variable among the different individual HPL used [14]. Osteogenic differentiation was assessed in 21/35 papers. The evaluation of the osteogenic differentiation by ALP activity was performed in 4 papers and showed some differences based on the cell type. In fact, while ASCs in HPL-supplemented culture showed a higher ALP activity than in FBS-supplemented ones [11,17], this was not seen for BMSCs [3,25]. Curiously, one study reported a higher ALP activity in HPL-cultured compared to FBS-cultured BMSCs. However, no significant differences in intracellular ALP activity between BMSCs and ASCs was noticed [11].
Expression of osteogenic markers was reported in 4 papers. Curiously, ALPL expression was markedly decreased in ASCs but slightly increased in BMSCs when cultured in HPL [25,37]. On the other hand, both BMSCs and ASCs differentiated in the presence of HPL overexpressed OCN and slightly downregulated RUNX2 compared to the same cells differentiated in FBS [11,25,35,37]. Further quantitative data, such as the quantification of stained cells or calcium deposition, resulted to be slightly superior in HPL-cultured compared to FBS-cultured cells [17,25].
Chondrogenic differentiation was reported in 11/35 papers. Among these, only one paper reported quantitative data and evidenced that ASCs cultured in HPL contained a lower level of GAGs compared to ASCs cultured in FBS [26]. Chondrogenic marker genes such as AGC1, COL9A2, and SOX9 were analyzed too in the same study and no differences in their expression were observed basing on the type of supplement [26].

Safety
Almost one third of the papers (10/35) investigated safety parameters. In particular, authors did not detect abnormal telomerase activity nor differences of the relative telomere length between HPL-and FBS-cultured cells [18,40]. Accordingly, senescence associated genes and β-galactosidase were expressed at the same level with both culture supplements [14,21,35,39]. Moreover, no signs of chromosomal alterations and tumorigenesis were retrieved after culturing cells with HPL [23,28,31].

Paracrine activity of MSCs
The data regarding the secretory profile reported in 6 papers were analyzed. Among all the investigated proteins, only two (vascular endothelial growth factor-VEGF and bFGF) were analyzed in at least two papers. Particularly, the level of VEGF was increased from 2 to 3.5 times in conditioned medium containing HPL compared to FBS [31,40,43]. Conversely, one paper reported a decrease of VEGF in HPL-conditioned medium [33] compared to FBS-medium. Other two papers reported a slight increase of bFGF levels in HPL-cultured cells [40,43].

Immunosuppressive abilities
Approximately one-fourth of the studies (9/35) investigated whether HPL supplementation affected the immunosuppressive abilities of MSCs (Table 1). Among the variety of assays performed, the mixed lymphocyte reaction (MLR) and the T Cells proliferation inhibition were the most represented. The outcomes of the MLR assay showed that the supplementation with HPL rather than FBS did not affect the ability of MSCs to inhibit the proliferation of peripheral blood mononuclear cells (PBMCs). Similarly, the inhibitory role on T cells proliferation exerted by MSCs was not affected by HPL or FBS. One study only showed a decrease of the inhibitory effects of BMSCs on PBMCs proliferation when cultured with HPL-supplemented medium in comparison to FBS-supplemented one [34]. Moreover, another study reported a superior immunosuppressive ability against natural killer (NK) cells proliferation of BMSCs supplemented with FBS than HPL [30].

Influence of the HPL production procedure on primary outcomes
Various protocols were reported for home-made HPL production in 31 out of 35 articles (Table 4). One third of the papers reported to use whole blood to produce HPL (10/31), whereas the source material for the other articles was PRP (7/31), platelet apheresis (7/31), or buffy coat (7/31). The number of donors ranged from 4 to 400, suggesting a great variability of protocols among laboratories. Platelet lysis was performed in different ways. Most studies (20/31) used less than three freeze/thaw cycles, while 11 between three and five freeze/thaw cycles. Sonication steps were reported in 2 records [29,37]. A key step in the HPL production is the fibrin removal to prevent clot formation, thus avoiding the need to add porcine heparin in the culture medium. This step was achieved through the incubation with CaCl 2 in combination or not with glass beads only in 2 papers that consistently did not include heparin in the final culture medium [17,20]. All the other 29 records did not remove fibrin during the HPL production and 4 of them did not use heparin in the final culture medium [29,33,34,37]. Considering the large heterogeneity in term of protocols for HPL production, we investigated a possible correlation between the variables of production and the primary outcome defined as DT. Among the variables considered, the source material, the procedure for platelet lysis, and the addition of heparin in the final culture medium were the ones reporting enough data to be compared. In particular, the source material and the heparin addition had no effect on the average DT (Fig. 5A, C). It was not possible to evaluate the influence of whole blood as platelet source since the DT was reported only in one paper. Conversely, the DT seemed to be influenced by the number of cycles used for platelet lysis. In facts, papers applying less than 3 freeze/thaw cycles reported a higher DT, corresponding to slower cell proliferation, compared to papers employing 3 or more freeze/thaw cycles, albeit this trend was not statistically significant (Fig. 5B). Moreover, we did not note any clustering related to the type of cells. Notably, one record was not considered since the DT values reported were outliers [33].

Risk of bias across studies
Overall, the 35 articles included showed moderate quality. A complete list indicating the specific bias for each study is reported in Additional file 3. The main bias retrieved were: Category 1-sample size and processing: Sample size of almost each study included more than 3 donors. Four studies included less than 3 donors and three studies did not specify the sample size. Sample processing was performed in the same way regardless of the experimental group, with only some bias concerning the time points considered related to the different rate in reaching cell confluence among FBS and HPL. Category 2-suitability of the detection assay: As for the detection methods, 6 papers investigated cell proliferation by measuring cell metabolic activities by assays such as MTT or MTS, which was considered as a bias since cell metabolic activity cannot be considered as a direct measure of cell number. Category 3-reproducibility and consistency of described methods: A consistent lack in the description of protocols was found. Indeed, 25 papers out of 35 did not report detailed information in their material and method section to make the procedures reproducible by third parties. As for the experimental setting, the most common bias was related to the normalization of gene expression analysis. In fact, stability tests on reference genes were generally lacking which somehow penalizes the consistency of the data reported. Category 4-completeness of the results. Another common bias retrieved in 16 papers was the lack of some results that were anticipated in the method section. Lastly, data from 22 papers were partially reported as raw number, thus preventing the comparison among studies.

Discussion
Considering the potential risks of zoonoses transmission and anaphylactoid reactions related to FBS after injection in human patients [4], xenogeneic-free alternatives for MSC expansion intended for medicinal products are strongly encouraged by European Medicine Agency (EMA) (EMEA/CHMP/410869/2006). Among all the alternatives proposed, the use of HPL is progressively increasing among the cell factories. However, to date its use for research purposes is still rare as hampered, among other reasons, by the lack of definitive evidences regarding its superiority over FBS and the lack of standardized protocols defining its use. For these reasons, we systematically reviewed the literature related to in vitro studies involving MSCs from adipose tissue and bone marrow, highlighting similarities and differences deriving from the use of HPL and FBS. The results of this systematic review first highlight that both BMSCs and ASCs proliferate faster when cultured in HPL-supplemented media than in FBS, regardless of its concentration. Specifically, both BMSCs and ASCs have a lower DT and a higher PD when cultured in HPL than FBS, allowing to obtain a higher amount of MSCs compared to FBS thus reducing the culture period. Importantly, it is clearly demonstrated that both cell types maintain unaltered features, such as the immunophenotype, the differentiation potential, the safety, and the immunosuppressive abilities regardless the type of supplement used. Conversely, other features such as the clonogenic potential, the pluripotency gene expression, and the secretion of active factors are slightly modified by the presence of the HPL. Moreover, both cell types are smaller and more spindle-shaped when cultured in HPL. Finally, the data analyzed show that the cell growth is positively influenced when platelet lysis is performed with at least three freeze/thaw cycles, providing an indication about the importance of the procedure chosen for the platelet lysis step to increase the cell performance.
Usually, the frequency of MSCs ranges from 1 to 10% of the total nucleated cells isolated [44]. Here, we summarize that the supplementation with HPL has no influence on this frequency in both ASCs and BMSCs compared to FBS as evidenced by the same number of colonies. However, colonies are heterogeneous and display different sizes and cell distribution. Indeed, given the higher DT and the lower PD in HPL-cultured cells, the studies analyzed showed that the colonies obtained in the presence of HPL are more densely populated and bigger than FBScultured cells. Moreover, HPL induces the increase of the expression of genes involved in the regulation of pluripotency and self-renewal pathways, such as NANOG, OCT4, and SOX2, suggesting that it enhances the stem potential [45].
One of the main issues related to MSC-based therapies is the possibility of tumor formation in a long-term evaluation [46]. Albeit the increase of the proliferation rate and the stem potential, no differences were observed between HPL-and FBS-cultured cells in terms of cell senescence, karyotype, and telomerase activity among the records analyzed here. However, this particular aspect is still poorly investigated when using HPL supplement, therefore additional evaluation would be needed to confirm the safety of MSCs supplemented with HPL and expanded ex vivo.
Our results evidenced that ASCs and BMSCs cultured with HPL secrete a higher level of VEGF and bFGF compared to counterparts cultured with FBS. This particular data is interesting as an increasing number of evidences highlights that MSCs exert their therapeutic potential mainly through the paracrine effect of extracellular vesicles and biologically-active secreted factors that stimulate survival and proliferation of resident cells, promote angiogenesis and modulate the cell immune response [47]. In particular, the initial growth of the new vascular network driven by pro-angiogenic factors represents a crucial point to obtain a successful tissue regeneration [48]. Therefore, an increase of the secretion of VEGF in MSCs supplemented with HPL represents a great advantage compared to classic procedure of FBS-based culture.
Another fundamental feature of MSCs is their ability to evade host immune system. The culture of ASCs or BMSCs with HPL maintained the ability to suppress the proliferation of T cells at comparable levels of FBScultured cells. The ability to bypass host immune system is mainly due the low expression level of MHC-ABC on their surface, making them invisible to NK cells. Moreover, MSCs do not express MHC-DR proteins, thus impeding the identification by CD4 + T cells [46]. Here, we observed that the expression of histocompatibility complex molecules is not modified when MSCs are cultured in HPL compared to FBS, therefore the shifting toward this human supplement does not hamper MSC therapy success. Along with this, MSCs possess a broad immunoregulatory ability and can suppress immune cells involved in innate and adaptive immune systems [49]. This unique immunologic abilities were firstly discovered in 2002 by demonstrating that the proliferation of leukocytes was suppressed when co-cultured with BMSCs in a dose-dependent manner [50]. Thus, MSCs exert their effect through both paracrine and cell-to-cell action [49], secreting different soluble factors some of which inhibit T cell proliferation and activation, such as hepatocyte growth factor (HGF), transforming growth factor (TGF)-β1, IL-10, and prostaglandin E2 [46]. Moreover, the lack of expression of co-stimulatory molecules, such as CD80 and CD86, is maintained in HPL-cultured cells as well as with FBS and does not provide a secondary signal during MSC-T cell interaction, thus suppressing T cell activation [49].
Albeit HPL reduces the immunologic risks linked to FBS, its human origin arises other issues, such as the transmission of blood-borne viruses, when it is obtained as a pooled product from multiple donors [28]. Autologous HPL can represent an alternative to avoid these side effects. However, autologous HPL could bring also some drawbacks. Indeed, each batch of autologous HPL            can have different characteristics due to the features of individual donors, such as age, gender, and thrombocythemia. As evidenced in two retrieved papers, the differences among individual batches of HPL can result in significantly different proliferation rate, cell morphology, and adipogenic differentiation level [14,15]. Hence, albeit cell response to autologous HPL is still superior compared to the culture with FBS, the donor-related variability of individual HPL batches may have an impact on the outcome of cell therapies, reducing the reproducibility of the procedure. One of the main hurdles to the shift from FBS to HPL is represented by the lack of recommendations on the protocol of production of HPL together with the concentration of use in MSC cultures. Indeed, its composition and bioactivity could possibly be influenced by several factors of the production process, such as source material, platelet count, lysis process, and other [40]. Up to now, a common consensus on HPL preparation and analysis is still missing, therefore the American Association of Blood Banks and the International Society of Cell Therapy are attempting to overcome this issue by establishing quality control criteria and by standardizing the manufacturing process [40].
Different donor features, such as gender and age, could influence the final HPL product and, subsequently, the MSC response. For example, female donors could present antibodies raised against human leukocyte antigens and human neutrophil antigens from previous pregnancies, thus creating unexpected cell responses [2]. Moreover, since aging is a systemic process, platelets could also be influenced by donor age. Indeed, it was demonstrated that MSC proliferation was significantly higher when cells were cultured with HPL obtained from donors < 35 years compared to HPL obtained from donors > 45 years [51]. This same study highlighted also that HPL from older donors increased the activity of senescence-associated β-galactosidase and decreased osteogenic differentiation